Lysine 219 participates in NADPH specificity in a flavin-containing monooxygenase from Saccharomyces cerevisiae

The flavin-containing monooxygenase from Saccharomyces cerevisiae (yFMO) us es NADPH and O-2 to oxidize thiol containing substrates such as GSH and the reby generates the oxidizing potential for the ER. The enzyme uses NADPH 12 times more efficiently than NADH. Amino acid sequence analysis suggests th at Lys 219 and/or Lys 227 may act as counterions to the 2' phosphate of NAD PH and to help determine the preference for pyridine nucleotides. Site dire cted mutations show that Lys 219 makes the greater contribution to cosubstr ate recognition. Conversion of Lys 219 to Ala reduces NADPH dependent activ ity 90-fold, but has no effect on NADH-dependent activity. Conversion of Ly s 227 to Ala reduces NADPH-dependent activity fivefold and NADH-dependent a ctivity threefold. Dissociation constants for NADP(+) to oxidized yFMO were measured spectroscopically. K-d is 12 mu M for the wild-type enzyme and 24 3 mu M for the K219A mutant, consistent with the role of Lys 219 in pyridin e nucleotide binding. (C) 1999 Academic Press.